O⁴‐Alkylated‐2‐Deoxyuridine Repair by O⁶‐Alkylguanine DNA Alkyltransferase is Augmented by a C5‐Fluorine Modification

Abstract

Oligonucleotides containing various adducts, including ethyl, benzyl, 4-hydroxybutyl and 7-hydroxyheptyl groups, at the O atom of 5-fluoro-O -alkyl-2'-deoxyuridine were prepared by solid-phase synthesis. UV thermal denaturation studies demonstrated that these modifications destabilised the duplex by approximately 10 °C, relative to the control containing 5-fluoro-2'-deoxyuridine. Circular dichroism spectroscopy revealed that these modified duplexes all adopted a B-form DNA structure. O -Alkylguanine DNA alkyltransferase (AGT) from humans (hAGT) was most efficient at repair of the 5-fluoro-O -benzyl-2'-deoxyuridine adduct, whereas the thymidine analogue was refractory to repair. The Escherichia coli AGT variant (OGT) was also efficient at removing O -ethyl and benzyl adducts of 5-fluoro-2-deoxyuridine. Computational assessment of N1-methyl analogues of the O -alkylated nucleobases revealed that the C5-fluorine modification had an influence on reducing the electron density of the O -C bond, relative to thymine (C5-methyl) and uracil (C5-hydrogen). These results reveal the positive influence of the C5-fluorine atom on the repair of larger O -alkyl adducts to expand knowledge of the range of substrates able to be repaired by AGT.

Influence of C5‐Substituents on Repair of O4‐Methyl Adducts of Pyrimidines by O6‐Alkylguanine DNA Alkyltransferases

“…Surprisingly, the repair of these C5‐halogenated adducts by hAGT is still more efficient than that of T ‐ O 4 Me (Table 1), demonstrating accommodation of these groups within the active site. In spite of their size, the susceptibility of the O 4 ‐Cα bond fostered by the C5‐halogen substituent (as previously observed with FdU ‐ O 4 Me ) and interactions within the active site seem to be playing a role in the repair observed by hAGT [32] …”

“…As it was previously observed by our group that the presence of fluorine at the C5 position of uracil greatly enhances the lability of the O 4 ‐alkyl group during oligonucleotide deprotection, similar mild treatments were employed for this series of modifications [31,32] . For solid‐phase synthesis of the oligonucleotides with phosphoramidites 2 a‐d and 6 a ‐ d , commercially available mild deprotection 3′‐ O ‐phosphoramidites were used with phenoxyacetic anhydride as the capping reagent [32] .…”

“…[31,32] For solid-phase synthesis of the oligonucleotides with phosphoramidites 2 a-d and 6 a-d, commercially available mild deprotection 3'-O-phosphoramidites were used with phenoxyacetic anhydride as the capping reagent. [32] Mild cleavage from the solid support and deprotection were accomplished with an anhydrous solution of potassium carbonate in methanol (0.05 M) for four hours at 21°C under gentle rocking, followed by neutralization of excess base with an equimolar amount of acetic acid. [33,35] were purified by SAX-HPLC and characterized by ESI-MS.…”

“…In spite of their size, the susceptibility of the O 4 -Cα bond fostered by the C5-halogen substituent (as previously observed with FdU-O 4 Me) and interactions within the active site seem to be playing a role in the repair observed by hAGT. [32] The hOGT protein used in this study is an engineered chimera of human AGT and E.coli OGT, where residues 139-159 from the human protein are replaced with their OGT counterparts with the additional S140P modification for protein solubility. [41] Repair of the O 4 -methyl series by hOGT followed a similar trend as the hAGT with the various C5-modifications following the order F > Cl > I > Br > CF 3 .…”

“…Starting from the commercially available nucleosides 5-chloro-2'-deoxyuridine, 5-bromo-2'-deoxyuridine, 5-iodo-2'-deoxyuridine and 5-trifluoromethyl-2'-deoxyuridine a dimethoxytrityl protective group is first introduced at the 5'-hydroxyl to give nucleosides (1 a-d) following standard procedures. [32] Then, the 3'-hydroxyl group is protected as a tert-butyldimethylsilyl ether to afford nucleosides (3 a-d). These nucleosides were then transformed to the C4-triazolyl convertible intermediates (4 a-d) by the slow addition of 1,2,4-triazole, triethylamine and phosphoryl chloride while stirring at 0°C.…”